Hematopoietic stem cells are cells which combine the potential of multi-differentiation and autoreproduction. The potential of autoreproduction is most important for hematopoiesis in order that blood cells not become exhausted over the course of a lifetime. With respect to the ability of the hematopoietic stem cells to multi-differentiate, as shown in FIG. 1, stem cells differentiate into myeloid stem cells and lymphatic stem cells, and these further differentiate into platelets, (mature) erythrocytes, granulocytes, monocytes, or the like from the myeloid stem cells, while blood cells such as T cells or B cells or the like are produced from the lymphatic stem cells.
Blood cells have a lifetime and are consumed in accordance with a variety of physiological needs, so that it is necessary that the blood cells be appropriately replenished by differentiation from stem cells. In patients suffering from, for example, acute myelogenous leukemia, there are irregularities in the differentiated functional blood cells themselves, as well as in the stem cell differentiation, so that the replenishment of functional red blood cells, white blood cells, platelets and the like is difficult. The transplantation of hematopoietic stem cells offers a treatment method for such blood diseases which does not have the side effects of chemotherapy and results in the recovery of functional hematopoiesis through the differentiation and regeneration of these cells. However, despite these advantages, the acquisition of stem cells is difficult, as they are almost all distributed in the bone marrow. Although placental blood and umbilical cord blood are comparatively enriched in stem cells, so that a less invasive method of obtaining them is possible, they can be used only in childbirth. On the other hand, in the peripheral blood which can be obtained from a donor in the least invasive way, the amounts of stem cells are further reduced which makes the peripheral blood less practical.
Furthermore, in transplanted blood cells containing stem cells, immunological rejection reaction, termed graft-versus-host disease (GVHD), may be induced when the HLA type of the patient and donor do not match. Accordingly, in order to conduct effective and safe transplantation of stem cells, it is necessary to obtain a stem cell sample from which lymphocyte fractions which give rise to GVHD are removed.
Furthermore, if pure stem cells could be isolated, they could be effectively stimulated and expanded using cytokines. Consequently, stem cell isolation could contribute to the development of stem cell banks in which such cells were stored for later use.
On the other hand, in concert with the recent development in genetic manipulation techniques, efforts have been made to conduct prenatal gene diagnosis for fetal nucleated cells. Fetal nucleated cells for diagnostic use which are currently clinically employed, are collected through invasive methods such as amniocentesis, chorionic villous sampling, and fetal blood collection, and these carry the risk of infection and amniorrhexis. It is conventionally known that fetal cells are admixed in the maternal blood, and the use of maternal peripheral blood to obtain fetal nucleated cells as a non-invasive collection has been considered; however, nucleated red blood cells (NRBC) being likely fetal cells are contained in the maternal peripheral blood in very small amounts, being only 1 in 105-107 of the total nucleated cells in the peripheral blood, so that the key to genetic diagnosis of fetal cells has been how to concentrate, separate, or identify such cells.
In addition, it is known that gene diagnosis is effective for the therapy of leukemia For example, since leukemia includes various types such as myeloid type in which hematopoietic cells themselves are pathologic or other type in which peripheral lymphocytes or monocytes become malignant, it is necessary to identify the type of leukemia for determining optimal dosing or therapeutic regimen. Moreover, genetic examination is needed to know the stage of differentiation of blood cells in which a carcinogenic factor is induced because the detection of the stage in which carcinogenic cells occur would contribute not only to the treatment of leukemia but also to clinically important matters such as prevention or recurrence of cancer. In such an examination of leukemia, it is also necessary to simplify and make effective the gene diagnosis in each differentiation stage by selecting and purifying immature hematopoietic blood cells and proliferating and differentiating them with cytokines.
The present inventors have conducted research which focused on the specific interactions between carbohydrates and other biological substances, and have filed a patent application on a method for selectively binding lectins, carbohydrate-specific proteins, to a solid support, such as a dish or the like, covered with synthetic glycoconjugate polymers including carbohydrate moieties (Japanese Patent Application No. Hei 8-59695).
On the other hand, as is shown in FIG. 1, hematocytes derived from hematopoietic stem cells express a variety of carbohydrate chains on the cell surface in accordance with the maturation thereof. In FIG. 1, the designation “Gal” indicates galactose, “Glu” indicates glucose, and “Lac” indicates lactose (Glu-Gal). In the patent application referred to above, it is disclosed that mature human erythrocytes expressing galactose are preferentially attached to the surface of the substrate covered with the glycoconjugate polymer including galactose, via a lectin (Allo-A) which recognizes galactose.
The present inventors have now thoroughly explored a control method for blood cell immobilization on a solid support covered with glycoconjugate polymers via lectins, and have discovered that by means of the incubation temperature or the amount of lectins added, a specific system of interactions among the cells and/or the carbohydrate moieties in the polymers and the lectins can be produced; the present invention was arrived at on this basis.